Chris Osburn

The ability to continuously monitor fecal bacteria concentrations in nearshore waters through field sensing has the potential to transform the way in which bacteria-driven public health risks are anticipated, mitigated, and managed by allowing for near real-time detection and the creation of high-quality datasets from which forecast models can be developed. Advances in freshwater monitoring reveal that fecal contamination can be predicted using data collected via high-frequency water quality sondes, but additional research is needed to extend these frameworks to coastal waters. We propose to observe water quality conditions every 15 minutes in Bald Head Creek, North Carolina, a tributary of the Cape Fear River, using a multiparameter sonde (YSI EXO2). The sonde will include sensors to monitor conductivity, temperature, dissolved oxygen, pH, turbidity, total algae (phycocyanin, phycoerythrin, and chlorophyll), fluorescent dissolved organic matter, tryptophan-like fluorescence, and water depth. In addition to observing water quality variables, we will analyze creek water samples for fecal indicator bacteria, antibiotic resistant bacteria, amino acids, and particulate and dissolved organic carbon isotopic signatures across four intensive field campaigns. Data collected via this project will be used to develop an innovative observation and machine learning modeling framework for predicting fecal contamination at high frequencies. Insights gained through the project will be shared with local and federal partners (e.g., Village of Bald Head Island, NC Coastal Federation, FDA Division of Seafood Science).

Osburn and group will collaborate with the project team to discover sources of dissolved organic nitrogen (DON), which is an emerging source of nutrient pollution contributing to the over-enrichment of nitrogen in NC??????????????????s coastal rivers and estuaries in the Chowan, Neuse, and Tar-Pamlico River basins. His group will receive samples collected by research partners and citizen scientists. Samples will be filtered in the lab and split for absorbance, fluorescence measurements and an aliquot archived for future use. Measurement corrections for instrument will be applied and corrected fluorescence data modeled for DON sources with FluorMod. The proposed project will have a series of benchmarks and milestones laid out to provide a means of tracking success. FluorMod will be applied iteratively as samples arrive after collection residual fluorescence not captured by FluorMod contains information about a watershed or river basin that can provide further insight to developing a new source for the model. Samples will be divided into training, validation, and test subsets which allows us to utilize some observations for discovering new signals to expand FluorMod and some observations for validating FluorMod??????????????????s efficacy. Osburn and group will participate in project meetings and assist with report and manuscript preparations.

Livestock operations generate fecal waste and manure management is an essential aspect of livestock production. Local and state regulations mandate permitting, training, design specifications, and stream vegetation buffers between livestock operations and surface waters. The scale of pork production has increased to meet consumer demand and as production facilities have grown, communities have heightened their concern about the environmental impact of pork operations. Pork producers have worked effectively to reduce their overall water usage, land use, and the carbon footprint of farming operations (National Pork Board, 2018, Thoma et al. 2011). Responsible environmental farm management has become a business necessity for pork producers in the US and producers have affirmed their obligation to manage pork production operations in a manner that protects natural ecosystems and public health. Watersheds, however, generally support multiple types of livestock operations and human dwellings. Each livestock enterprise and residential community is a potential source of fecal waste in surface waters. Fecal waste in surface waters is generally referred to as non-point source contamination. In reality, all fecal waste has a vertebrate animal origin and the species of origin varies with adjacent land-use practices. Monitoring programs established to protect public health have traditionally relied on the culture or detection of fecal coliforms, total coliforms or Enterococcus bacteria in water samples as indicators of fecal contamination in surface waters. These enteric organisms are non-specific indicators of the presence of fecal waste but do not attribute contamination to specific animal hosts. The detection of host-specific enteric organisms, such as Bacteroidales spp. and genetic assays focused on detecting these microbial species have been developed as alternatives to non-host specific indicator organism detection methods (Harwood et al. 2009). All vertebrates release cells from their gastrointestinal tract in their feces. These cells contain mitochondrial DNA (mtDNA), a routine aspect of forensic investigation that can be applied to identify the animal hosts associated with fecal waste (Caldwell et al. 2007). The detection of mtDNA is highly host specific. If mtDNA is detected in a water sample, the vertebrate animal associated with that mtDNA can be determined. Initial studies, however, lacked sensitivity (Caldwell et al. 2009). When we initially developed and tested these assays, at times, fecal waste was present in a stream but not detected. In studies supported by the National Pork Board, we refined these initial mtDNA assays by adapting the use of new Droplet digital PCR technology (BioRad Inc., California, USA), which markedly enhanced the sensitivity of the assay for identifying the presence of host mtDNA in surface waters. New primers and probes were designed, and the assay proved both sensitive and specific. Our studies confirmed the presence of fecal contamination in Stockinghead Creek in Duplin County, NC and documented that the fecal contamination in the creek originates from at least four species, cattle, humans, poultry and swine. This proposal focuses on addressing concerns about the origin of fecal waste in surface waters in other North Carolina livestock intensive watersheds. Specific objectives include: 1) Attributing the source of fecal contamination in NC surface waters in Duplin and Sampson County, NC watersheds ; and 2) Responding to concerns about fecal contamination.

All livestock operations generate fecal waste and manure management is an essential aspect of pork production. Regulations mandate permitting, training, design specifications, and stream vegetation buffers. As the scale of pork production has increased to meet consumer demand communities have heightened their concern about the environmental impact of pork operations. Pork producers are working to reduce their overall water usage, land use and carbon footprint of farming operations (National Pork Board, 2018). Responsible environmental farm management has become a necessity and pork producers in the US have affirmed their obligation to manage pork production operations in a manner that protects natural ecosystems and public health. Watersheds generally support multiple types of livestock operations and human dwellings. Each is a potential source of fecal waste in surface waters. However, when fecal material is detected it is often swine farms that are the target of concern. Fecal waste in surface waters is generally referred to as non-point source contamination. In reality, all fecal waste has a vertebrate animal origin and the species of origin varies with adjacent land-use practices. Monitoring programs established to protect public health have traditionally relied on the culture or detection of enteric bacteria in water samples, which do not differentiate contamination from animals hosts. Genetic assays focused on detecting microbial species have also been developed as an alternative to these culture-based methods. Prior studies by the NC Division of Environmental Quality (DEQ) and our laboratories have noted the presence of fecal coliform contamination in Stockinghead Creek, Duplin County, NC. Additional microbial-based studies conducted by other laboratories suggested pigs were the origin of the fecal waste and public reaction targeted the swine operations in the watershed. All vertebrates release cells from their gastrointestinal tract in their feces. These cells contain mitochondrial DNA (mtDNA), a routine aspect of forensic investigation that can be applied to identify the animal hosts associated with fecal waste. Unlike the majority of molecular microbial-based approaches noted above, mtDNA detection is highly specific. If mtDNA is detected in a water sample, the vertebrate animal associated with that mtDNA can be determined. However, an initial real-time polymerase chain reaction assay we developed lacked sensitivity. In our initial Pork Board funded studies we refined the mtDNA assay for classifying the species of origin of fecal contamination in surface waters by adapting the use of new droplet digital PCR technology. New primers and probes were designed, and the assay proved both sensitive and specific. Our studies confirmed the presence of fecal contamination in Stockinghead Creek and documented that the fecal contamination originates from at least three species, cattle, humans and swine. This renewal is requested to continue source tracking for fecal waste in the Stockinghead Creek Watershed. In addition, we will work with DEQ and test samples they collect in the Stockinghead Creek Watershed. Specific objectives include: 1) Broadening the species detected with the Droplet Digital mtDNA assay; 2) Conducting parallel sampling with DEQ to develop a more comprehensive assessment of fecal contamination in the watershed; and 3) Comparing and contrasting results obtained with the mtDNA assays with two bacterial-based assays for identifying the animal hosts contributing to fecal contamination. Water is one of our most limiting resources. The proposed studies address the environmental mitigation technology and practice objectives stated by the National Pork Board Sustainability Committee. By facilitating accurate detection of fecal waste in rivers and streams the proposed studies support the objective of the Producer/public health Workplace Safety Committee focused on reducing exposure to zoonotic pathogens. The regulatory necessity of ensuring that water resources are free of fecal waste sometimes pl

All livestock operations generate fecal waste and manure management is an essential aspect of pork production. Regulations mandate permitting, training, design specifications, soil testing and livestock operation stream vegetation buffers. As the scale of pork production has increased to meet consumer demand communities have heightened their concern about the environmental impact of pork operations. Pork producers are actively working to reduce their overall water usage, land use and the carbon foot-print of their farming operations. Responsible environmental farm management has become an inherent necessity to maintain the sustainability of the pork industry and pork producers in the US have affirmed their obligation to safeguard our natural resources and manage pork production operations in a manner that protects natural environments and public health. The proposed studies support environmental management of pork production and address community concerns by facilitating accurate detection and effective attribution of the origin of fecal waste in surface waters and groundwater.

Freshwater mussel populations throughout North America have declined precipitously during the last three decades. Captive propagation and release of the captive reared stock has become an integral component of efforts to mitigate the decline and augment the reproductive capacity of remaining populations. Freshwater mussels are reared in captivity using two alternative approaches that focus on different approaches to facilitating the metamorphosis of juveniles. Either host-fish are used to support the metamorphosis of the larval stage or the larvae are reared in vitro in petri dishes and the metamorphosis is nutritionally supported with culture media. The initial survival of in vitro reared mussels is poor and their initial growth lags behind that of host-fish reared animals. Our limited understanding of the nutritional needs of freshwater mussels and how specific components of their diet contribute to their nutritional health impedes our ability to sustain them in captivity. In addition, declines noted in some free-ranging populations appear to be associated with poor nutrition. We propose studies to further inform our understanding of the role of different food-web resources in the diets of freshwater mussels. Specific objectives include: 1) Examining the role of pollen in the diet of freshwater mussels; 2) Assessing the role of detritus in freshwater mussel diets; 3) Quantifying the filtration ability of selected freshwater mussels species; 4) Preliminary studies of diet and freshwater mussel nutritional health; and 5) Refining procedures for characterizing the chemical composition of freshwater mussel shells.

A nitrogen (N)-based Total Maximum Daily Load (TMDL) was implemented for the N-sensitive Neuse River Estuary (NRE), beginning in 1996, as an approach to reduce eutrophication and control the recurring large phytoplankton blooms and associated water quality and habitat degradation (harmful algal blooms, hypoxia/anoxia, fish kills). The TMDL mandated a 30% reduction in total N loading to the system and requires chlorophyll-a (Chl a) concentrations to be below 40 ?????????g/L for 90% of samples collected (Session Laws 1995, Section 572). While reductions in dissolved inorganic N (DIN) loading have taken place (Lebo et al., 2012), nuisance phytoplankton blooms and high Chl a measurements persist (Paerl et al., 2009). The persistence of Chl a measurements above the 40 ?????????g/L threshold could be a reflection of the changing forms of N. Despite a documented reduction in DIN, dissolved organic N (DON) has been increasing in the NRE over the past 20 years (Lebo et al., 2012). We hypothesize the increasing abundance of DON, specifically those sources related to the rapidly growing poultry operations and urbanization as reflected by septic outflow in rural, coastal NC, is sustaining the observed Chl a exceedances. The proposed study will conduct DON nutrient addition bioassays coupled with environmental surveys conducted in the NRE to research the link between changing N-sources, primarily as DON sources, to phytoplankton primary production and community composition. If results suggest these sources (poultry litter leachate, septic outflow, natural wetlands runoff) of DON stimulate phytoplankton primary productivity or select for harmful algal bloom taxa, then these bioreactive DON sources should be targeted in addition to DIN in efforts to control and mitigate the negative impacts of eutrophication on anthropogenically impacted N-sensitive coastal systems like the NRE.

Overview: Chromophoric dissolved organic matter (CDOM) is an important fraction of the marine carbon cycle that controls most light absorption and many photochemical and biological processes in the ocean. Despite its importance, the chemical basis for the formation of oceanic CDOM remains unclear. Laboratory studies support the paradigm that bacterial transformation of phytoplankton particulate organic matter (POM) and DOM produces the humic-like CDOM signals observed in the deep ocean. However, prior studies of oceanic CDOM using absorbance and fluorescence fit an electronic interaction (EI) model of intramolecular charge transfer (CT) reactions between donor and acceptor molecules common to partially-oxidized terrestrial molecules (e.g., lignin) found in humic substances. This proposal will test the hypothesis that phytoplankton and bacteria provide a source of donors (e.g., aromatic amino acids) and acceptors (quinones) which are microbially-transformed and linked, enabling CT contacts between them and creating oceanic CDOM. Hotspots for the formation of planktonic CDOM may be marine aggregates of phytoplankton detritus (marine snow). We will systematically study phytoplankton growth including marine snow formation, using roller bottles as a laboratory set-up that favors the formation of marine snow. A new technique of measuring base-extracted POM (BEPOM) absorbance and fluorescence will assist in testing fit of planktonic CDOM to the EI model, supplemented with measurement of its probable chemical precursors, thus explaining the production of CDOM in the ocean by linking the optics and chemistry of planktonic CDOM formation. Determining the time course and extent of phytoplankton POM and DOM transformation by heterotrophic bacterial (enzymatic hydrolysis) during the same phytoplankton growth experiments will provide an in-depth understanding as to how bacterial transformation of marine snow-associated planktonic organic matter drives CDOM production throughout the ocean. Intellectual Merit: This work represents a mechanistic study that will improve our understanding of the role of phytoplankton as the major source of CDOM in the open ocean. The key merit of this proposal is testing the fit of the EI model to planktonic CDOM via examination of: 1) its fluorescent quantum yields, 2) wavelength dependence of its fluorescence emission, and 3) alteration of its absorbance and fluorescence after borohydride reduction. These results will determine if intramolecular charge transfer occurs between reduced aromatics derived from amino acids and microbially-sourced quinones. A second merit is quantifying the importance of aggregate formation for oceanic CDOM formation. One implication of these results is that biomolecules in phytoplankton as on land must undergo a microbially-mediated transformation prior to developing chemical structures that give rise to CDOM?s optical properties. Broader Impacts: The importance of planktonic CDOM is not restricted to oceanography and the knowledge gained in this project will transfer to limnology and aquatic biogeochemistry as a whole. This study will provide unequivocal evidence for the remote sensing community that the CDOM spectra in the open ocean (and some lakes) are derived from phytoplankton. Each PI will utilize data gained from their respective research in classroom instruction and student mentoring. Beyond the classroom, we will work the NCSU Science House and the Nature Research Center at the North Carolina Science Museum of Natural Sciences to connect this important research topic with the general public. Using results from the work proposed, we will develop an interactive multimedia module for public display entitled, ?Light and Life in the Ocean,? at the NC Museum of Natural Sciences. Visitors to the display will be able to modify light via computer interface and learn effects of light on the ocean ecosystem. This module will be complemented with demonstrations and presentations at the UNC-CH Science Expo, a yearly event that introduces over 8000 members of the general public to scientific research taking place at UNC. As is possible, the PIs will conduct live feeds of shipboard and laboratory activities via Skype to classes at elementary schools in the Raleigh area so students can ?Ask an Oceanographer? questions regarding the experience and importance of conducting oceanographic research at sea and in the laboratory.

The aim of this proposal is to optimize algorithms that integrate optical and chemical information of dissolved organic matter (DOM) based on proxies for the prediction of its flux from marshes to coastal waters through estuaries. Land use and climate are important drivers that strongly influence the transport and fate of coastal wetland DOM offshore and these transitional areas have also been recognized recently as important sinks in the global carbon pool, commonly referred to as "blue carbon." Coastal wetlands in Louisiana, (e.g., marsh-estuarine complexes such as Barataria Bay) show clear decreasing gradients of DOM quantified as dissolved organic carbon (DOC) and dissolved lignin that suggest loss of blue carbon from the marshes to the more estuarine bays and subsequent export to coastal waters. The overarching hypothesis of this study is that changes to DOM chemistry within the marsh-estuarine complex imparts seasonal variability in the quantity and quality of DOM exported to the coastal ocean. This hypothesis is important to test because DOM reactivity to sunlight and/or bacteria regenerates mineral nutrients and CO2. Light-absorbing DOM (i.e., CDOM) also is a function of its chemistry, allowing CDOM retrievals from remote sensing reflectances to predict DOM quantity. The next step in the advancement of our understanding of the terrestrial-marine linkage?and the potential loss of blue carbon from coastal wetlands?is to predict DOM quality synoptically with remote sensing. We propose to test our hypothesis by combining co-varying chemical (dissolved lignin, stable isotopes) and optical (fluorescence) biomarkers that can deconvolve complex mixtures of DOM sources, along with optical measurements relatable to remote sensing observations. We plan to conduct seasonal (spring and fall) field campaigns in the Barataria Bay to modify existing algorithms for the VIIRS sensor, building on prior NASA-funded CDOM work by our team as part of the OCB and NACP programs. Coastal wetlands are economic powerhouses, supporting local fisheries productivity, recreation, aesthetics ? and their protection is a critical coastal management issue. Along the Gulf Coast of the United States, coastal wetlands are some of the most critically sensitive ecosystems under threat from anthropogenic and climatic stressors, particularly along the Louisiana coast. The Barataria Basin contains swamps, fresh, brackish, and saline marshes and bayous, in addition to the estuarine Bay proper, and serves as an ideal study site. The region has been heavily impacted from human modification such as subsidence, reduction of marshes from eustatic sea level rise, hurricanes, loss of resupply of materials from rivers due to channelization and construction of artificial levees. This proposal specifically addresses Item 3.2 Theme 2 related to carbon dynamics along the terrestrial-aquatic interface. Together, DOM chemical and optical properties can distinguish between terrestrial, marsh, and estuarine vegetation. Substantial variability exists in the sea-to-air fluxes of CO2 that likely is linked to the fate of blue carbon DOM in coastal waters as contributed by coastal wetlands. Remote sensing estimates of these sources obviously would improve our understanding of the role that coastal wetlands play in the contribution of continental margin systems to global carbon budgets, especially with respect to changing patterns of climate and land use. Quantifying the exchange of DOC between wetlands and shelf regions is critical to do now, especially in light of the impending rise of sea level will alter these fluxes; particularly in regions like Louisiana where relative sea level rise (RSLR) and wetland loss rates are considerably higher than other regions in the country. This information gap will be addressed with the proposed work.

Extreme weather events -- such as the record rainfall and massive flooding recently experienced along the southeast and mid-Atlantic US coasts from Hurricane Matthew -- are becoming more frequent and occurring with greater intensity. The importance of these events for coastal ocean biogeochemistry remain largely unknown because while events such as tropical storms are ephemeral, last perhaps a few days to a week, their effects on coastal environments last perhaps for multiple years to decades. During a storm, it can be very difficult (and life-threatening) to sample, yet the response of coastal ecosystems to an event can be observed and provide critical information and understanding regarding the spatial extent and magnitude of material fluxes of bioactive elements such as carbon (C) and nitrogen (N) to the coastal ocean. This is the purpose of our RAPID proposal. Amidst the chaos of such extreme events and their resulting effects on natural and human ecosystems, clear imperative biogeochemical questions emerge regarding these ????????????????hot moments???????????????. For example, 1) How do coastal C and N budgets respond to floodwaters from tropical storms and hurricanes? 2) What is the biological and photochemical reactivity of this material? The first question can be answered via a relatively short, yet intense, period of observations, such as we propose here. The second question, which has potential teleconnections to climate in terms of CO2 fluxes, food web responses, and other ecosystems processes, requires a longer duration study. However, Question 2 could be answered by further study of samples collected during a short and intense period of sampling proposed to answer Question 1. In this RAPID proposal, we aim to improve our understanding of how estuaries and coastal systems respond to extreme events by measuring carbon and nutrient (N and P) loading into the Neuse River Estuary-Pamlico Sound (NRE-PS) coastal ecosystem. The NRE-PS is the urgent study site to examine resulting effects of Hurricane Matthew on coastal environments because it is downstream of the most intense flooding that occurred. Furthermore, it is the focus of a long-term monitoring program in that system, the Neuse River Estuary Modeling and Monitoring Program (ModMon), which was initiated in 1993. That date is significant because it preceded a recent rise in Atlantic tropical cyclone activity and has been able to capture the biogeochemical and ecological effects of major storms that have impacted the NC coast, including Hurricanes Fran (1996), Floyd (1999), Isabel (2003), Irene (2011). Incorporating Matthew will enable a comparison of these storms to the historic record. We propose to estimate fluxes and reservoirs of key constituents such as dissolved inorganic carbon (DIC), dissolved and particulate organic carbon and nitrogen (DOC, POC, DON, PON), and N and P nutrients, as well as chlorophyll biomass and pigment analyses. What this information will provide is a critical snapshot of the material fluxes into this coastal environment resulting from a major tropical storm and provide a comparison across spatiotemporal dimensions of the NRE-PS and other coastal environments such as the Chesapeake Bay, Mississippi River plume, etc.